Various archaeological and historical evidence shows that the marginal area of the Negev desert of southern Israel enjoyed great agricultural prosperity in the Byzantine period (4th–7th centuries CE). Among the different types of agricultural installations are pigeon towers, which were built near the fields to produce fertilizer to enrich the nutrient-poor desert soils. Such extensive specialized agriculture practice was much less applied in the Negev in the successive Early Islamic period in the mid-7th century. Here we recovered in situ pigeon bones from five pigeon towers in the Negev, applied multiple characterization methods (FTIR, grinding curve, and C/N ratio) to estimate the preservation of bones, and achieved absolute dating for the abandonment of the towers. The obtained dates indicate rapid decline of agricultural activities in the second half of the 6th century CE and beginning of the 7th century. These findings, together with other evidence for Byzantine decline of agricultural hinterland and urban dysfunction of the settlements, suggest that the farming activities in the Negev declined in the Late Byzantine period (550–640 CE) and support the hypothesis that climatic-driven causes were the main trigger for the eventual cultural-societal decline of the Negev region.