Campylobacter (C.) jejuni and C. coli are responsible for food poisoning in humans. Laying hens may host the bacteria usually without developing symptoms. The aims of this paper were to evaluate the incidence of C. jejuni and C. coli infection in laying hen flocks housed in different rearing systems, the plasma levels of two welfare indicators (corticosterone and interleukin 6, IL-6) and the antimicrobial resistance of the detected Campylobacter strains. Two different flocks (1 and 2) from cage (A), barn (B) and aviary (C) farms were investigated. The highest (p < 0.05) levels of IL-6 were detected in laying hens housed in aviaries. A similar trend emerged in corticosterone level, although differences were found between C1 and C2. C. jejuni and C. coli were identified in 43.5% and 38.9% of birds, respectively. In total, 14 out of 177 (7.9%) hens simultaneously hosted C. jejuni and C. coli.C. jejuni was prevalently detected in hens housed in barns (B1: 53.3%; B2: 46.7%) and aviaries (C1: 34.6%; C2: 86.7%). Conversely, laying hens housed in cages were significantly exposed to infection of C. coli (A1: 41.9%; A2: 80%) while, regarding barns and aviaries, a significant prevalence emerged only in flocks B2 (40%) and C1 (54.8%). Simultaneous infection was statistically significant in barn B1 (36.7%). Antibiotic resistance was mainly detected among C. coli strains, and it was most frequent for fluoroquinolones and tetracycline. Multidrug resistance was also found in C. jejuni (19.7%) and C. coli (17.5%) strains. Based on the results of this study, we recommend increasing biosecurity and hygienic measures to manage hen flocks.