Fungal endophytes may contribute to plant health and disease protection, yet little is known about their various roles in alfalfa. Also, endophytes from several plant species produce novel antimicrobial compounds that may be useful clinically. We isolated endophytic fungi from over 50 samples from six locations in Minnesota and Idaho; both healthy and diseased alfalfa displaying wilt symptoms were collected. Sequence and BLAST analysis of the ITS region revealed a variety of genera but were dominated by Alternaria spp. Of 20 isolates tested, 17 were positive for antimicrobial production against at least one test organism using streak plate assays against 12 bacterial and fungal strains including both human and alfalfa pathogens. Of these, three Alternaria isolates were grown for three weeks in solid substrate rice fermentation cultures that were subsequently extracted with ethyl acetate, dried and resuspended in methanol. Disc diffusion assays using 1 mg of crude extracts produced clear zones of inhibition against two Gram - and six Gram + bacteria, including Staphylococcus aureus and Clavibacter michiganensis. Fungistatic and bacteriostatic activity was seen for Candida albicans and Pseudomonas syringae, respectively.