You've made a good start certainly. Here are a few pointers to consider:

1. Your input file "contig1 ac346... etc" wasn't in a code block, so the tab characters don't render properly ...

2. I'm fairly sure others would agree that declaring a scalar with the same name as a hash (or an array) is ambiguous and asking for trouble. In your particular case, Perl does seem to do what you intended, however, later in the future you may extend this script and all of a sudden it breaks.

    my $med = median(@{$organisms{$organism}});  #bad form, masks %med
+ declared in outer scope. Use a different name for this variable, eg.
+ $med_calculated.
    $med{$organism} = $med;
[download]

(Also, same goes for your $number assignment section)

3. When submitting scripts onto PM, use the <DATA> handle for your source data - it makes things a little more portable, and easier for others to reproduce what your seeing, and then offer assistance or a solution.

4. I'm not sure why you're calculating the median of $organisms{$organism} before its been populated with anything... maybe you need another loop after you've finished reading in your source data...?

5. Your headline appears to have 5 columns, but your data printing loop:-

foreach $organism (sort {$number{$a} <=> $number{$b}} keys %organisms)
+{
    print "$organism:\t$number{$organism}\t$organisms{$organism}\t$med
+{$organism}\n" ;
}
[download]

only has 4 values. something quite innocent like this is often an "oh yeh, i know thats wrong, but I'll fix it later". Make things easier for yourself. The k.i.s.s. acronym is a very wise one...

6. As you've discovered, the median function takes a list as it's input: print median(1, 100, 101);   # 100

since Perl's hashes and arrays can only ever hold scalars (or references), something isn't quite right there with the the median calculation... A good place to start would be to break down what you've got in 1 step, into 3 or 4... This also makes debugging easier as you can insert warn $varible_name statements to confirm they contain the values you are expecting them to.

use strict;
use warnings;

use Acme::Tools;

my (%count, %organisms, %med, %number);
my ($contig, $accession, $organism, $eval, $con_length, $map_length);

#my $ref_filelist = $ARGV[0];
#open(FILELIST, $ref_filelist )
#   or die "Could not open Reference filelist...($!)";
 
print "Organism\tFrequency\tMedian_Eval\tMedian_Contig_Length\tMedian_
+Mapped_Length\n"; 

#while (<FILELIST>){
while (<DATA>){
    ( $contig, $accession, $organism, $eval, $con_length, $map_length 
+) = split ( '\t',);

    #my $median = $eval[($#eval / 2)];
    my $med_calculated = median(@{$organisms{$organism}});  
    $med{$organism} = $med_calculated;

    #$organisms{$organism} = $eval;
    my $number_calculated = ++$count{$organism};    
    $number{$organism} = $number_calculated;
}

foreach $organism (sort {$number{$a} <=> $number{$b}} keys %organisms)
+{
    print "$organism:\t$number{$organism}\t$organisms{$organism}\t$med
+{$organism}\n" ;
}


__DATA__
contig1    AC344    organism1    1e-1    122    45
contig1    AC344    organism1    1e-2    122    45
contig1    AC346    organism2    1e-102    122    46
contig1    Ac346    organism2    1e-100    122    46
contig1    Ac346    organism2    1e-114    122    46
contig1    Ac346    organism2    1e-111    122    46
contig2    NC333    organism3    1e-2    155    90
contig3    NC444    organism4    1    188    50
contig3    NC444    organism4    12    188    50
[download]