The growing demand for alternative sources of non-animal proteins has stimulated research in this area. Mushrooms show potential in the innovation of plant-based food products. In this study, the aim was to develop prototype fish fillets analogues from Pleurotus ostreatus mushrooms applying enzymatic treatment (β-glucanase and transglutaminase-TG). A Plackett-Burman 20 experimental design was used to optimize forty variables. Oat flour (OF) exerted a positive effect on the hardness and gumminess texture parameters but a negative effect on cohesiveness and resilience. Soy protein isolate (SPI) exhibited a positive effect on elasticity, gumminess and chewiness, while acacia gum had a negative effect on elasticity, cohesiveness and resilience. After sensory analysis the assay with 1% cassava starch, 5% OF, 5% SPI, 0.1% transglutaminase (240 min/5 °C), 1% coconut oil, 1% soybean oil, 0.2% sodium tripolyphosphate, 0.6% β-glucanase (80 °C/10 min) and without β-glucanase inactivation was found to exhibit greater similarity to fish fillet. The classes hydrocarbons, alcohols and aldehydes are the predominant ones in aromatic profile analysis by chromatography and electronic nose. It is concluded that a mushroom-based analogue of fish fillet can be prepared using enzymatic treatment with TG.