Evaluation of environmental risk factors in the development of autism spectrum disorder (ASD) is needed for a more complete understanding of disease etiology and best approaches for prevention, diagnosis, and treatment. A pilot experiment in 54 children (n = 25 ASD, n = 29 controls; aged 12.4 ± 3.9 years) screened for 87 urinary mycotoxins via liquid chromatography-tandem mass spectrometry to assess current exposure. Zearalenone, zearalenone-4-glucoside, 3-acetyldeoxynivalenol, and altenuene were detected in 9/54 (20%) samples, most near the limit of detection. No mycotoxin/group of mycotoxins was associated with ASD-diagnosed children. To identify potential correlates of mycotoxin presence in urine, we further compared the nine subjects where a urinary mycotoxin was confirmed to the remaining 45 participants and found no difference based on the presence or absence of mycotoxin for age (t-test; p = 0.322), gender (Fisher's exact test; p = 0.456), exposure or not to selective serotonin reuptake inhibitors (Fisher's exact test; p = 0.367), or to other medications (Fisher's exact test; p = 1.00). While no positive association was found, more sophisticated sample preparation techniques and instrumentation, coupled with selectivity for a smaller group of mycotoxins, could improve sensitivity and detection. Further, broadening sampling to in utero (mothers) and newborn-toddler years would cover additional exposure windows.