How can I delete a newline if it is the last character in a file?

perl -pe 'chomp if eof' filename >filename2

or, to edit the file in place:

perl -pi -e 'chomp if eof' filename

^{[Editor's note: -pi -e was originally -pie, but, as noted by several commenters and explained by @hvd, the latter doesn't work.]}

This was described as a 'perl blasphemy' on the awk website I saw.

But, in a test, it worked.

You can take advantage of the fact that shell command substitutions remove trailing newline characters:

Simple form that works in bash, ksh, zsh:

printf %s "$(< in.txt)" > out.txt

Portable (POSIX-compliant) alternative (slightly less efficient):

printf %s "$(cat in.txt)" > out.txt

Note:

If in.txt ends with multiple newline characters, the command substitution removes all of them.^{Thanks, Sparhawk} (It doesn't remove whitespace characters other than trailing newlines.)
Since this approach reads the entire input file into memory, it is only advisable for smaller files.
printf %s ensures that no newline is appended to the output (it is the POSIX-compliant alternative to the nonstandard echo -n; see http://pubs.opengroup.org/onlinepubs/009696799/utilities/echo.html and https://unix.stackexchange.com/a/65819)

A guide to the other answers:

If Perl is available, go for the accepted answer - it is simple and memory-efficient (doesn't read the whole input file at once).
Otherwise, consider ghostdog74's Awk answer - it's obscure, but also memory-efficient; a more readable equivalent (POSIX-compliant) is:
awk 'NR > 1 { print prev } { prev=$0 } END { ORS=""; print }' in.txt
Printing is delayed by one line so that the final line can be handled in the END block, where it is printed without a trailing \n due to setting the output-record separator (OFS) to an empty string.
If you want a verbose, but fast and robust solution that truly edits in-place (as opposed to creating a temp. file that then replaces the original), consider jrockway's Perl script.

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