Did you notice you have a print outside the if as well as inside it? As well as the single quotes and wrong comparison operators that the others have mentioned. There's no reason for you to slurp the file and then iterate through the lines, you can read it a line at a time. This ought to do what you want:

my $input = $ARGV[0];

my $count = -127.5;
my $value1 = $count - 2.5;
my $value2 = $count + 2.5;

open(INPUT, "$input") || die "Oops!: Can't open file: $!\n";
while (<INPUT>) {
    my $checkvalue = substr($_, 0, 6);
    print "$checkvalue $value1 $value2\n" if $checkvalue > $value1 and
+ $checkvalue <= $value2;
}
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