Prosopis laevigata(mesquite; Fabaceae) forms fertility islands in soils of semi-arid lands where microbial diversity concentrates in response to the accumulation of resources in the soil beneath individual plants, promoting organic matter decomposition and nutrient cycling. This phenomenon provides suitable conditions for the proliferation of key edaphic elements such as fungi and mites. Mite-fungal interactions are central for our understanding of nutrient cycling processes in resource-limited arid food webs; yet, no information is available about fertility islands in semi-arid lands. Thus, we aimed to determine in vitro fungal-based feeding preferences and molecular gut content of the oribatid mite speciesZygoribatulacf.floridanaandScheloribatescf.laevigatus, which are abundant under the canopy ofP. laevigatain an intertropical semi-arid zone in Central Mexico. Our results on the gut content analysis of these oribatid species resulted in the ITS-based identification of the following fungi:Aspergillus homomorphus,Beauveria bassiana,Filobasidiumsp.,Mortierellasp.,Roussoellasp.,Saccharomyces cerevisiae, Sclerotiniaceae sp. andTriparticalcarsp. Furthermore, under laboratory conditions both oribatid mite species exhibited feeding preferences on melanized fungi, such asCladosporiumspp., whereasA. homomorphusandFusarium penzigiwere avoided. Our findings indicated that the analyzed oribatid mite species have similar feeding preferences for melanized fungi, which might suggest resource partitioning and a degree of preference, explaining the coexistence of both oribatid species.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10493-023-00790-7.