In Konya, Türkiye-a semi-arid agricultural region where 90% of water consumption is for irrigation. Farmers in this region have relied on the “de facto” reuse of secondary treated wastewater for irrigation. This reclaimed water can contain residual pollutants, including antibiotics that can harm the ecosystem. Türkiye is one of the most antibiotic-consuming countries, raising growing concerns about antibiotic transfer through food chains when reclaimed water is used for irrigation. This field-scale study investigated the occurrence, fate, and uptake of antibiotics from reclaimed wastewater into lettuce irrigated with five different water sources: wastewater (WW), groundwater (GW), secondary clarified water (SCW), ultrafiltration effluent (UF), and multi-media filter with ultraviolet (MMF/UV) effluent. Antibiotic levels in irrigation water and lettuce were quantified using liquid chromatography–tandem mass spectrometry (LC–MS/MS), which identified 20 antibiotics and four metabolites. The highest concentrations were observed in the WW samples, and the lowest in the UF samples. Clarithromycin (CLA, 362.4–933.8 ng/L) dominated all water types. Antibiotic accumulation was greater in leaves (651.52–1045.10 ng/g) than in roots (386.36–811.87 ng/g). Monte Carlo simulation was applied to evaluate health risks and parameter sensitivities. For children, hazard quotient (HQ) values ranged from 3.23 × 10−3 to 7.33 × 10−3 both values were lower in adults. All measured HQ values were below threshold limits. Thus, consuming lettuce irrigated with reclaimed water containing antibiotics poses negligible risk to humans under current assumption.