#!/usr/bin/perl

$ENV{CLUSTALDIR} = 'C:\\\\\\\\\\\\\\\\\\;
use Bio::Tools::Run::Alignment::Clustalw;

my $file = <>; # Get file name (fasta file) from command prompt.

#  Build a clustalw alignment factory
my $factory = Bio::Tools::Run::Alignment::Clustalw->new(-matrix => 'BL
+OSUM',-outfile=>'out', -output =>'clustalw'); 
my $ktuple = 2;
$factory->ktuple($ktuple);

my $inseq = Bio::SeqIO->new(
                    -file=> "<$file",
                    -format=> 'fasta'
                   );
my $seq;
my @seq_array;
while ($seq = $inseq->next_seq) {
    push(@seq_array, $seq);
}
# Now we do the actual alignment.
my $seq_array_ref = \@seq_array;
my $aln = $factory->align($seq_array_ref);

#constarcting the Tree
my $alnio = Bio::AlignIO->new(-file => 'out', -format=>'clustalw');

my $dfactory = Bio::Tree::DistanceFactory->new(-method => 'NJ');

my $stats = Bio::Align::ProteinStatistics->new;

my $treeout = Bio::TreeIO->new(-format => 'newick');

while( my $aln = $alnio->next_aln ) 
{
 my $mat = $stats->distance(-method => 'Kimura',
                            -align  => $aln);
 my $tree = $dfactory->make_tree($mat);
 $treeout->write_tree($tree);
}
my $outfile = 'C:\Users\Ioulia\Desktop\outree.tre';
my $treeout = Bio::TreeIO->new(-format => 'newick', -file => ">$outfil
+e");
exit;
[download]

I think you need to create the $treeout object with -file first so that it uses it as the stream for the write_tree method

my $outfile = 'C:\Users\AnyUser\Desktop\outree.tre';
my $treeout = Bio::TreeIO->new(
    -format => 'newick', 
    -file   => ">$outfile" );

while( my $aln = $alnio->next_aln ) 
{
  my $mat = $stats->distance(-method => 'Kimura',
                            -align  => $aln);
  my $tree = $dfactory->make_tree($mat);
  $treeout->write_tree($tree);
}
exit;
[download]

poj

You are absolutely right! Bless you my friend!