Although attention on crayfish diseases has recently proliferated, the focus is mainly on a single host-parasite relationship rather than analyzing the entire mycoflora probably due to the fact that (I) some diseases are occasional (pathogens of which are of difficult collection) and (2) economic impact is more relevant in aquaculture (i.e., a controlled environment where only few parasites occur) than in natural conditions. Contrary to this viewpoint, fungal ectoparasites assume a great importance since they are integral components in shaping community, ecosystem structure, and energy flow, Here we described the fungal species co-occurrence patterns (species segregation/aggregation) using null models comparing the occurrence frequencies of ectosymbiots through host individuals with those expected by chance. Non-indigenous crayfish species like Procambarus clarkii allow to observe phenomena of competitive exclusion (segregation) or mutualism (aggregation) amongst fungi. We analyzed the fungal species occurrence on 86 host crayfish monthly collected from June 2007 to June 2008 in the Lake Trasimeno (Central Italy). A total of 29 fungal taxa were detected. Taxa showed a particular fungal assemblage with a great species variation depending on sampling month and crayfish sex, size, and body region. In particular fungal species showed different occurrence patterns in both richness and segregation/aggregation. The fungal community shows a non-random structure not in all cases, with the majority of tests indicating segregation, not aggregation of ectosymbiontic species on hosts. The non-random patterns of fungal co-occurrence in some cases suggest that some temporally or spatially variable factors are responsible for the establishment of the mycoflora community assemblage. We also detected temporal differences in fungal co-occurrences patterns. Regarding seasonal samples, the Same analyses showed a competitive structure only in the sub-communities found on pleopodal coxae. In addition, analyses considering crayfish dimension evidenced non-random co-occurrence patterns only in crayfish hosts with a cephalothorax length larger than 45 mm. Our results demonstrated that, depending on sampling month and crayfish sex, size, and body region, well structured fungal assemblages on crayfish hosts can alternate with random fungal assemblages. Whenever non-randomness of fungal co-occurrence was detected, it hinted segregation, suggesting the lack of co-evolutive phenomena of mutualism, and favoring a competitive exclusion among the mycoflora species.