The glasshouse study was conducted with the objectives of (i) investigating the effect of soil moisture variations on the control efficiency of glyphosate on windmill grass (Chloris truncata R.Br.), common sowthistle (Sonchus oleraceus L.), and flaxleaf fleabane [Conyza bonariensis (L.) Cronq.], (ii) evaluating the tolerance of tested weed species under soil moisture variations, and (iii) determining the morphological and physiological characteristics of these species to partially explain herbicide tolerance under periods of reduced soil moisture availability (RSM). The species’ tolerance to glyphosate increased significantly under reduced soil moisture availability (p < 0.001). The lethal dose to cause herbicide injury or biomass reduction by 50% (LD50) and 80% (LD80) in relation to untreated control for water-stressed plants [i.e., moderate soil moisture availability (MSM) and RSM] was significantly higher than that of plants grown under high soil moisture availability (HSM). The tolerance factor (TF) for C. truncata, S. oleraceus, and C. bonariensis, in terms of biomass reduction under RSM, was 2.6, 2.4, and 2.6, respectively, as compared to HSM. The results showed that the glyphosate sensitivity, especially at the sub-lethal rates, of the three weed species under study decreased as soil moisture availability reduced (p < 0.01). Overall glyphosate efficacy, in relation to the recommended rate, was unaffected, except for C. truncata; the weed survived the highest tested glyphosate rate [750 g active ingredient (a.i.) ha−1] under RSM. There was significant interaction between weed species and soil moisture regimes for weed morpho-physiological traits (p < 0.001), with reduced soil moisture having a more influential impact on the growth of C. bonariensis and S. oleraceus compared to C. truncata. Changes in the leaf characteristics, such as increased leaf thickness, higher leaf chlorophyll content, reduced leaf area, and limited stomatal activity for all the tested weed species under MSM and RSM in relation to HSM, partially explain the tolerance of species to glyphosate at sublethal rates.