Measurement of gas concentrations constitutes basic knowledge for the computation of emissions from livestock buildings. Although it is well known that hot climate conditions increase gas emissions, in the literature the relation between gas concentrations from open barns and animal-related parameters has not been investigated yet. This study aimed at filling this gap by evaluating daily gas concentrations within an open-sided barn in hot Mediterranean climate. The influence of microclimatic parameters (MC) and cow behavior and barn management (CBBM) were evaluated for ammonia (NH3), methane (CH4), and carbon dioxide (CO2) concentrations. Results showed that both MC and CBBM affected concentrations of NH3 (p < 0.02), CH4 (p < 0.001), and CO2 (p < 0.001). Higher values of NH3 concentration were detected during the cleaning of the floor by a tractor with scraper, whereas the lowest NH3 concentrations were recorded during animal lying behavior. Measured values of CO2 and CH4 were highly correlated (C = 0.87–0.89) due to the same sources of production (i.e., digestion and respiration). The different management of the cooling systems during the two observation periods reduced significantly CH4 concentrations in the barn when the cooling system in the feeding area was switched off. Based on methodological choices due to the specific barn typology, parameters related to animals can provide information on the variation of gas concentrations in the barn environment in hot climate conditions.