My .htaccess file has got the current setting:

Options -Indexes

RewriteEngine On
RewriteCond %{REQUEST_FILENAME} !-f
RewriteCond %{REQUEST_FILENAME} !-d
RewriteRule ^(.*)$ $1\.php

After I enter mydomain.com/file it's ok, however when I access mydomain.com/file/ I recieve error 500.

There is no folder known as "file", this issue seems with any path.

Try adding -MultiViews for your Options. Whether it still breaks, consider the apache error_log, you will get the entire explaination of what is failing.