Shualim Rockshelter in the Central Negev Highlands bears stratified deposits of three Upper Paleolithic (UP) occupation phases belonging to the Ahmarian cultural entity and an Initial Upper Paleolithic (IUP) industry. Microarchaeological investigations were undertaken to identify site formation processes and detect evidence of human activities, including investigating combustion features that were identified during field excavations. Our microarchaeological approach utilized Fourier-transform infrared spectroscopy (FTIR) and Fourier-transform infrared micro-spectroscopy (micro-FTIR), quantification of ash pseudomorphs, phytolith analysis and sediment micromorphology. Based on multiproxy microarchaeological signals, we identify the in situ use of fire including two combustion features from an Upper and Lower Ahmarian occupation phase. Ash pseudomorphs and gypsum neoformation indicate that woody taxa including Tamarix sp. were utilized as fuel sources, while seed coats resembling Brassicaceae suggest that human occupation during the upper Ahmarian phase took place during the winter season. The widespread occurrence of carbonated hydroxyapatite minerals and microscopic bone fragments reflects human utilization of faunal resources on-site. We also undertake a spatial assessment of the nature and degree of taphonomic processes and identify areas within the rockshelter that preserve intact archaeological contexts and abundant microarchaeological signals of local human activity. Our microarchaeological investigations provide a high-resolution window into the nature of human activities on-site and add to our understanding of the occupation dynamics of successive UP groups in the arid environment of the Negev desert.