The paper focuses on the application of ultrasonic energy in textile laundering. In recent years, there has been an increasing interest in ultrasonic energy application in textile industry; however, the effect of ultrasonic laundering on the thermophysiological properties of knitted fabrics has not been studied yet. This study was conducted by using polylactic acid (PLA), cotton, polyethylene terephthalate (PET), and poly acrylic (PAC) fibres containing yarns and their blends. Knitted fabrics, single pique, were made from these yarns by using weft knitting machine. The fabrics were washed ten times for 15 and 60 minutes under 40 °C by using conventional and ultrasonic washing methods. The main aim was to determine the effect of washing methods on the thermophysiological properties of the fabrics. It is also aimed to analyse and evaluate the thermophysiological properties of the PLA fabrics. The incorporation of 100 % PLA and cotton/PLA yarns into single pique knitted fabrics has been attempted to produce for the first time and studied their thermal comfort properties. The results show that the washing processes have a critical importance for the tested fabrics in terms of thermal conductivity, thermal resistance, thermal absorbtivity, water vapour permeability, and heat loss. It has been also demonstrated that the fabric cleaning by using ultrasonic method enhanced the properties of tested fabrics such as thermal conductivity and % recovery. It was also noted that 15 minutes ultrasonically washed fabrics had significantly lower thermal resistance as compared to conventionally washed fabrics.