Many freshwater mussel species are critically imperiled, and propagation is essential for species ‘recovery.’ Fungal contamination can negatively affect in vitro propagation of freshwater mussels; thus, we investigated methods of mitigating fungal contamination. Specifically, we tested the effect of medium replacement frequency and antifungal (Amphotericin B) concentrations on risk of fungal contamination and transformation success of two species, Alasmidonta heterodon and congener, Alasmidonta undulata. We observed a trend of increased contamination risk in treatment groups that received medium replacements every 1 or 2 days (vs. every 3 days), but this trend was not statistically significant. We found that contamination risk differed significantly across Amphotericin B concentrations. Although contamination severity reduced transformation success, we found no significant difference in transformation success based on medium replacement frequency. Amphotericin B concentration was negatively correlated with transformation success, suggesting toxicity at higher concentrations. Additionally, we identified the fungal pathogen (Candida parapsilosis), the first knowledge of this fungus during mussel culture. Our findings suggest that low contamination results in high probability for transformation success; however, there is a tradeoff in higher concentrations of Amphotericin B. This study offers new ideas for improving mussel propagation techniques that may better control fungal contamination and increase transformation success.