The use of agricultural chemicals has adversely affected soil health and the environment. Organic farming practices, particularly vermicompost (VC), are gaining attention for their potential to improve soil fertility and crop productivity. This study investigated VC rate applications on lettuce growth, yield, soil fertility, nutrient dynamics, enzyme activity, biological parameters, and biochemical aspects under greenhouse conditions in Samsun, Turkey during 2022–2023. Experimentally, VC was applied at rates of V1: 1%, V2: 2%, and V3: 4% w/w, with a control group without VC application, V0: 0% w/w. Batavia lettuce, which is sensitive to environmental conditions and nutrient deficiency, was subjected to these treatments in a randomized complete block design, replicated thrice. Results showed consistent improvements in plant dry weight across all VC treatments, with the 2% application rate (V2) yielding the highest increase in lettuce yield (56.43%). Soil pH varied across treatments, with V1 being slightly alkaline and V3 showing high electrical conductivity and increased nitrogen content. Phosphorus content increased in all treatments, while potassium varied, with V3 having the highest values. Soil enzyme activities increased with VC concentrations, with V3 showing the highest urease activity. Pearson correlations confirmed positive associations with growth parameters and soil enzymatic activity. These findings highlight vermicompost as a sustainable solution for lettuce production and soil improvement.