Acinetobacter baumannii is a leading cause of various hospital-acquired infections, including bacteraemia and pneumonia. This bacterium is notorious for its ability to persist on dry surfaces in clinical settings for extended periods, a factor believed to be critical in the development of nosocomial infections. However, molecular mechanisms behind A. baumannii's survival in such harsh environments are not yet fully understood. In this study, we used a transposon-directed insertion sequencing (TraDIS) based functional genomic approach to identify 41 genes that positively contribute to desiccation tolerance and 83 genes that have a detrimental effect on this tolerance in A. baumannii. Phenotypic analysis of 10 single-gene disruption mutants revealed that genes involved in global regulation (dksA and gacA), oxidative stress (katE), osmo-protection (otsB), DNA-binding histone-like protein (hns), and proteins related to cyclic di‐GMP signalling (efp) are important for survival during desiccation. Among these, dksA and efp had the most significant impact on desiccation survival. Conversely, disruption of the protease (lon) and the gene involved in two-component systems (ompR) enhanced desiccation tolerance in A. baumannii. Additionally, we demonstrated that DksA not only regulates many stress resistance genes but also newly identified desiccation tolerance proteins (dtpA and dtpB). These findings highlight that desiccation stress imposes multiple challenges on A. baumannii, with dksA playing a central regulatory role. Our results provide valuable insights into the genetic regulation and mechanisms underlying desiccation tolerance in this clinically significant pathogen.