The application of CO2 and diode lasers as an alternative thermal method for the non-chemical control of weeds is investigated. Under laboratory conditions, experiments were carried out on the interaction of laser light with mono- and dicotyledonous plants represented by Echinochloa crus-galli and Nicotiana tabacum. Two laser systems, three growth stages and different energy doses were used. The fresh masses of treated and untreated plants were measured and compared seven days following treatment. By fitting dose–response curves to the results it was possible to define a specific ED90 value for each combination of laser wavelength, growth stage and plant species. The ED90 intervals were classified by growth stage; increasingly higher doses were needed from seedling to second leaf growth stage and on to the third growth stage. For the treatments, the higher absorption of the CO2 laser wavelength yielded a higher efficiency than the diode laser. The plant species N. tabacum was found to be more sensitive than E. crus-galli. The different characteristics of the absorption spectra and the growth habits of the plants were regarded as the main reasons for the different effects. Overall, the application of laser radiation as a weed control method showed promising results, especially in the area of effectiveness and energy efficiency for non-chemical weed control.