The two-spotted spider mite, Tetranychus urticae, is an important pest. This mite has recently become a serious problem due to the continuous use of acaricides, resulting in resistance. Thus, there is a need to identify alternative control methods to suppress spider mite populations. We screened pathogenic fungi for the control of spider mites using multiple tools to assess virulence and fitness under various environmental conditions. Twelve selected fungal isolates demonstrated the virulence against mites, and their thermotolerance, UV-B tolerance and cold tolerance varied by isolate. The most optimal fungi to control mites were selected based on results that took various tested environmental factors into consideration. Additionally, the antimicrobial activities of the selected fungi were assessed to explore the potential for the dual control of not only mites but also plant pathogens. Antimicrobial activities against both the bacterium Bacillus cereus and the fungus Botrytis cinerea were observed simultaneously in 8 fungal isolates. These results suggested that the fungal isolates Lecanicillium attenuatum 4-1 and Purpureocillium lilacinum 2R-4-6 can be used in an eco-friendly control program against the two-spotted spider mite and Beauveria bassiana s.l. 2R-4-5 and Metarhizium anisopliae s.l. 4-2 would be powerful candidates for the dual control of pests and diseases.