Macrophomina phaseolina (Tassi) Goid., the causal agent of charcoal rot disease of soybean, is capable of causing disease in more than 500 other commercially important plants. This fungus produces several secondary metabolites in culture, including (-)-botryodiplodin, phaseolinone and mellein. Given that independent fungal isolates may differ in mycotoxin and secondary metabolite production, we examined a collection of 89 independent M. phaseolina isolates from soybean plants with charcoal rot disease using LC-MS/MS analysis of culture filtrates. In addition to (-)-botryodiplodin and mellein, four previously unreported metabolites were observed in >19% of cultures, including kojic acid (84.3% of cultures at 0.57–79.9 µg/L), moniliformin (61.8% of cultures at 0.011–12.9 µg/L), orsellinic acid (49.4% of cultures at 5.71–1960 µg/L) and cyclo[L-proline-L-tyrosine] (19.1% of cultures at 0.012–0.082 µg/L). In addition, nine previously unreported metabolites were observed at a substantially lower frequency (<5% of cultures), including cordycepin, emodin, endocrocin, citrinin, gliocladic acid, infectopyron, methylorsellinic acid, monocerin and N-benzoyl-L-phenylalanine. Further studies are needed to investigate the possible effects of these mycotoxins and metabolites on pathogenesis by M. phaseolina and on food and feed safety, if any of them contaminate the seeds of infected soybean plants.