Sinai's important geographical and strategic position is attracting researchers to explore opportunities to maximize exploitation of its treasures, especially in the area of sustainable development. One of the fields of exploitation is extracting valuable metals from low-grade ores using green technologies. In this study, we examined the possibility of microbial leaching of uranium (U) from a rock sample collected from Wadi Naseib, Sinai, Egypt. Twenty previously isolated and tentatively identified native microorganisms, 10 Streptomyces and 10 Aspergillus, were used to make U-bioleaching using cells (direct) and cell metabolites (indirect). The tested isolates showed variable U-bioleaching efficiencies and the highest results was attained via the indirect method (57.2 ± 9.2% and 83.6 ± 2.3%) using two isolates that were identified genotypically as Streptomyces sp. EGY1 and Aspergillus niveus EGY2 respectively. TEM images showed that cells of A. niveus EGY2 made biomineralization, biosorption and bioaccumulation of U. The present study revealed that neither high acid production nor high phosphatase activities guarantees a high U-bioleaching efficiency. Many factors affecting the process were also studied using A. niveus EGY2. The highest U-bioleaching efficiency (87.8 ± 8.7%) was attained using pH 9, 160 rpm of both culturing and bioleaching steps, rock particle size of above 700 µm and 1% pulp density. U was recovered from leach liquor after optimization experiments using NaOH and its concentration was 64.35%. Our study revealed that Aspergillus niveus EGY2 could be promising in future scaling-up studies and pilot trials using the tested rock sample.