(1) Background: In addition to the microclimate, host availability, and tick microbiota, soil environmental microorganisms can affect tick populations. This study aimed to (1) determine the presence and diversity of entomopathogenic fungi (EF) in forests, where ticks are abundant, and (2) estimate the effectiveness of the isolated EF strains against Ixodes ricinus. (2) Methods: EF were isolated using the trap insect method from soil collected from tick sites. A bioassay was used to estimate the effectiveness of EF against ticks. (3) Results: The presence of EF was found in all tested forest habitat types. A total of 53 strains belonging to the genera Metarhizium, Beauveria, and Isaria were isolated. All the six strains subjected to the bioassay showed potential efficacy against both adult and nymphal stages of I. ricinus; however, the strains differed in their effectiveness. The most effective isolate against I. ricinus was the soil environmental strain of Metarhizium anisopliae. (4) Conclusion: The study indicates that tick habitats can be the source of entomopathogenic fungi, which have a lethal effect on ticks, as demonstrated in preliminary laboratory tests with I. ricinus. However, for practical use, extensive field tests and further research on application methods and long-term effects are necessary to develop effective and sustainable tick management strategies.