Research interest in chiral pesticides has increased probably because enantiomers often exhibit different environmental fate and toxicity. An investigation into the enantiomer-specific bioactivity of chiral triticonazole enantiomers in agricultural systems revealed intriguing experimental and theoretical evidence. For nine of the phytopathogens studied (Rhizoctonia solani, Fusarium verticillioide, Botrytis cinerea (strawberry and tomato), Rhizoctonia cereali, Alternaria solani, Gibberella zeae, Sclerotinia sclerotiorum, and Pyricularia grisea), the fungicidal activity data showed (R)-triticonazole was 3.11–82.89 times more potent than the (S) enantiomer. Furthermore, (R)-triticonazole inhibited ergosterol biosynthesis and cell membrane synthesis 1.80–7.34 times higher than its antipode. Homology modeling and molecular docking studies suggested the distinct bioactivities of the enantiomers of triticonazole were probably due to their different binding modes and affinities to CYP51b. However, field studies demonstrated that (S)-triticonazole was more persistent than (R)-triticonazole in fruits and vegetables. The results showed that application of pure (R)-triticonazole, with its high bioactivity and relatively low resistance risk, instead of the racemate in agricultural management would reduce the application dosage required to eliminate carcinogenic mycotoxins and any environmental risks associated with this fungicide, yielding benefits in food safety and environmental protection.