While lettuce offers essential human nutrients, it also contains anti-nutrients, particularly nitrate (NO3−). The use of neem leaf extract as a natural nitrification inhibitor has proven itself promising to remediate lettuce tissue NO3− content. This study evaluated the effects of neem leaf extract on soil properties, soil nitrification, lettuce growth, yield, and NO3− content. Five nitrification inhibitor treatments were evaluated: (i) no inhibitor (control), (ii) nitrapyrin, and three rates of neem leaf extract based on the dry weight of the raw material: (iii) 1 g kg−1 soil (Neem1), (iv) 2 g kg−1 soil (Neem2), and (v) 4 g kg−1 soil (Neem4). Neem leaf extract generally increased soil concentrations: P (47.6–55.8 mg kg−1), K (45.8–62.7 mg kg−1), Ca (129–164 mg kg−1), and Mg (29.0–35.7 mg kg−1) compared with the control (50.6 mg P kg−1, 35.3 mg K kg−1, 123 mg Ca kg−1, and 24.8 mg Mg kg−1). Neem leaf extracts significantly increased soil NH4+–N concentrations (13.9–30.2 mg kg−1) and nitrification inhibition (12.5–70.5%), but significantly decreased soil NO3−–N concentrations (6.4–13.2 mg kg−1) and net nitrification rates (0.08–0.23 mg N kg−1 day−1) relative to the control (6.6 mg NH4+–N kg−1, 14.7 mg NO3−–N kg−1, 0.26 mg N kg−1 day−1, and 0% nitrification inhibition). The neem leaf extracts significantly decreased shoot fresh weight (13.5–43.1 g plant−1), shoot dry weight (0.84–3.91 g plant−1), and root dry weight (0.14–0.27 g plant−1) compared with the control (52.3 g shoot fresh weight plant−1, 5.36 g shoot dry weight plant−1, and 0.35 g root dry weight plant−1). The significant decreases in the lettuce biomass in the neem extract treatments paralleled the significant decreases in the shoot’s tissue NO3−–N contents and significant increases in tissue NH4+–N content and soil Al concentrations.