Fungal-derived food products align with sustainable food supply principles and offer a sustainable and nutritious option for consumers. Rhizomucor pusillus strain CBS 143028 has emerged as a candidate food ingredient. Fermentation of R. pusillus CBS 143028 results in a mycelium biomass mainly comprising fungal proteins, cell wall components, and micronutrients. Although R. pusillus has a history of safe use in the production of food enzymes and in traditional fermented foods, the fungal biomass obtained after fermentation of R. pusillus CBS 143028 is considered a novel food and requires a thorough safety assessment. To this end, a 90-day oral toxicity study was conducted in which Wistar rats (10/sex/group) were provided diets containing 0, 100,000, 200,000, or 300,000 ppm of R. pusillus mycelium. Standard toxicity study parameters as given in OECD Test Guideline 408 were examined. The mean achieved dosages of R. pusillus mycelium were 6398, 12,738, or 19,668 mg/kg body weight/day for males and 7235, 14,949, or 22,461 mg/kg body weight/day for females in the low-, mid-, and high-dose groups, respectively. Although statistically significant differences were reported, these effects were not considered biologically relevant or test article-related due to atypical values in the control groups, the lack of a dose response relationship, or were attributed to normal biological variation. Thus, the no-observed-adverse-effect level (NOAEL) was established at 300,000 ppm (corresponding to 19,668 and 22,461 mg/kg body weight/day for males and females, respectively), the highest concentration tested.