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, antifungal (Amphotericin B) concentrations, and antifungal addition method on risk of fungal contamination and transformation success of two species, Alasmidtonta heterodon and congener, Alasmidonta undulata. We found that contamination risk was higher in treatment groups that were replenished with Amphotericin B every one or two days (vs. every three days), but contamination risk was not affected by 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 of apparent toxicity 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.