An increasing amount of wastes associated with poultry raising in highly intensive farming environment has been the growing cause of concern. Huge, modern poultry farms face grave problems concerning the disposal of poultry litter, noxious odour emission, pollution of water, soil and air. The objective of the present research was to determine the effect of layer hens` age on quantitative and qualitative contamination of litter. The experimental material was constituted by the litter samples collected from three hen facilities (three age groups) in the farm of laying hens of Hy-line. The microbial analysis included: meso- and psychrophylic bacteria, bacteria from coli group, thermotolerant, proteolytic, etc. In the indoor sampling sites, there were also measured chosen microclimatic factors. The microbiological evaluation revealed the highest microbial contamination of litter, analyzing three age groups of laying hens, in the hen building K3 (birds aged 54 weeks), followed by K2 hen house (30-week-old birds) and the lowest in the K1 (6 weeks of age).