In this work, microwave irradiation was used to extract two dye plants (Quercus ithaburansis Decaisne and Rubia tinctorum L.) and to dye natural cotton. Using microwave (MW) energy, the extraction time for these plants and the dyeing time for organic cotton fabric were optimized. The colouring compounds were analysed in the extractions and dyed fabrics by reversed-phase high-performance liquid chromatographic method coupled with a photodiode array detector (RP-HPLC PAD). CIEL*a*b* spectrophotometer measured the colour characteristics of all the dyed fabrics. Scanning electron microscopy with energy dispersion spectroscopy (SEM-EDX) was used to image the dyed organic cotton fabrics. The colouristic and colourfastness properties of the dyed fabrics were investigated. According to the test results, it has been determined that the 5 minutes of extraction and dyeing time using with microwave irradiation method is very eligible. During these processes, energy and timesavings were achieved by eco-friendly MW irradiation and minimized cotton damage.