Rhizobium plays an important role in cultivation of legumes throughout nitrogen fixation process. In the present study, growth of alfalfa (Medicago sativa) was studied after treatment with Rhizobium sp. isolated from two different sites in Jordan. The effect of the two isolates (RS1 and RS2) and co-inoculation of the two isolates (RS1+RS2) was tested on alfalfa. Chlorophyll contents, relative water content, leaf area, shoot length, stem diameter and plant biomass (leaf, root and shoot dry weight) under growth chamber conditions (day/night temperatures of 25C; 16:8 h photoperiod) using different inoculum doses (10ml, 20ml and 40ml) was studied. The effect of Rhizobium isolates on alfalfa was compared with rhizosphere plant growth-promoting rhizobacteria (PGPR) collected from the same soil samples. The results indicated that RS1 at high dose (40ml) and RS2 at any dose significantly enhanced alfalfa growth in all measured parameters compared with control plants (without bacterial inoculation). RS2 was better than RS1 in most measured parameters even at low inoculum dose (10ml), whereas no significant differences between the two strains were found at high inoculum doses. Co-inoculation of the two isolates was less effective than each isolate alone. On the other hand, the PGPR showed a significant enhancement of alfalfa growth as they enhanced significantly all measured parameters compared to the control plants at all doses