Ras1 and Ras2 are two distinct Ras GTPases in Beauveria bassiana, an entomopathogenic fungus whose biocontrol potential against insect pests depends largely on virulence and multi-stress tolerance. The functions of both proteins were characterized for the first time by constructing dominant-active (GTP-bound) Ras1G19V and dominant-negative (GDP-bound) Ras1D126A and integrating them and normal Ras1 into wild type and ΔRas2 for a series of phenotypic and transcriptional analyses. The resultant mutants showed gradient changes of multiple phenotypes but little difference in conidial thermotolerance. Expression of Ras1D126A caused vigorous hyphal growth, severely defective conidiation, and increased tolerances to oxidation, cell wall disturbance, fungicide and UV-A/UV-B irradiations, but affected slightly germination, osmosensitivity and virulence. These phenotypes were antagonistically altered by mRas1G19V expressed in either wild type or ΔRas2, which was severely defective in conidial germination and hyphal growth and displayed intermediate changes in other mentioned phenotypes between paired mutants expressing Ras1G19V or Ras1D126A in wild type and ΔRas2. Their growth, UV tolerance or virulence was significantly correlated with cellular response to oxidation or cell wall disturbance. Transcriptional changes of 35 downstream effector genes involved in conidiation and multi-stress responses also related to most of the phenotypic changes among the mutants. Our findings highlight that Ras1 and Ras2 regulate differentially or antagonistically the germination, growth, conidiation, multi-stress tolerance and virulence of B. bassiana, thereby exerting profound effects on the fungal biocontrol potential.