Fungi, known for their adaptability, are valuable sources of enzymes, making them promising for biocatalyst discovery. This study explored Penicillium steckii, primarily recognized for secondary meta-bolite production, as a source of ene-reductases (ERs), which reduce α,β-unsaturated compounds. Eleven ER-encoding genes were iden-tified, and plasmids for Escherichia coli expression were generated. Six ERs (PsOYE1-6) were successfully produced and purified as soluble FMN-containing proteins. Sequence analysis classified them into Class II (PsOYE1, PsOYE4, PsOYE6), Class III (PsOYE2, PsOYE3), and Class V (PsOYE5) OYEs. All were active on p-benzo-quinone and maleimide, with varying activity on other substrates. Their pH optima ranged from 6 to 7, and they exhibited moderate thermostability (35-50 °C). PsOYE2 was crystallized, and its 2.3 Å structure revealed a stable dimer with a unique active site. PsOYE3, PsOYE4, and PsOYE5 were tested for R-carvone conversion and stereoselectivity, all favouring one diastereomer. These fungal ERs expand the enzymatic toolbox for biocatalysis, emphasizing the need for tailored strategies based on specific applications.