This research was aimed to determine: 1) the effect of diazotrophic bacteria application on nitrogen uptake, chlorophyll content, and rate of assimilation in shallot; 2). Selecting the best type of diazotrophic bacteria isolat to increase nitrogen uptake, chlorophyll content, and assimilation rate in shallot. The research was carried out in a screen house at an altitude of ± 110 meters above sea level and the Laboratory of Agronomy and Horticulture, Faculty of Agriculture, Jenderal Soedirman University, Purwokerto from February 2021 to April 2021. This study was arranged using a Randomized Completely Block Design (RCBD). The treatment in this research consisted of  I₁ = LCR3, I₂ = LAR5, I₃ = LBR1, I₄ = LAZ2, I₅ = LAZ3, I₆ = LCA1, I₇ = LAR3, I₈= LAA5, and I₉ = LAA4. There are 9 treatments and a control each repeated 3 times. The variables observed consisted of leaf area, shoot dry weight, root dry weight, total root length, root volume, root shoot ratio,chlorophyll content,  relative growth rate, net assimilation rate, and nitrogen uptake. The data obtained was analyzed using Analysis of Variance (ANOVA), if they were significantly different then further tested using DMRT  at the 5% level. The result showed that application of diazotroph bacteria had a significant effect on variables were net assimilation rate, and nitrogen uptake. The treatment of isolat I₂ (LAR5) resulted in the highest number of leaf area and nitrogen uptake variables.