Among edible mushrooms, Pleurotus spp. is widely cultivated, and interest is growing in reusing its spent substrate (SPS). SPS shows promise as an organic fertilizer to enhance soil fertility and physical properties; however, its impact on soil microbial activity remains underexplored. This study assessed raw SPS as an organic fertilizer for baby leaf lettuce, focusing on yield, nutritional quality, soil fertility, and soil enzymatic activity. The trial took place in a tunnel greenhouse across two crop cycles (Cycle 1: October–December; Cycle 2: January–March) using two lettuce varieties: Doge and Imperiale. Five fertilization treatments were applied: T0 (no fertilization), TMIN (mineral fertilizer NPK 35-7-4,75 kg ha⁻¹), T100+50 (35 kg ha⁻¹ N via SPS + 17.5 kg ha⁻¹ mineral N), T200 (70 kg ha⁻¹ N via SPS), and T200+50 (70 kg ha⁻¹ N via SPS + 17.5 kg ha⁻¹ mineral N). At harvest, aerial biomass and soil samples were collected to assess yield, plant mineral content, soil nutrients, and enzymatic activities (BGA, ArS, AcP, and AlkP). Fertilization significantly influenced yield: T0 and TMIN produced the highest yields (2.6 and 2.3 kg m⁻²), while T100+50 yielded the most among SPS treatments (2.0 kg m⁻²). T200 and T200+50 improved all enzymatic activities. Mineral profiles differed by variety: Imperiale had higher P, K, S, and Ca and the highest nitrate content under TMIN (37,232 mg kg⁻¹), while Doge absorbed more nitrates under SPS treatments (29,943 mg kg⁻¹). SPS did not affect soil P or K due to the short crop cycle. Overall, raw SPS enhanced soil enzyme activity and lettuce nutrient content, with T100+50 offering the best trade-off between soil health and yield. Future research will evaluate SPS use in other baby leaf crops.