Densely packed, motile bacteria can adopt collective states not seen in conventional, passive materials. These states remain in many ways mysterious, and their physical characterization can aid our understanding of natural bacterial colonies and biofilms as well as materials in general. Here, we overcome challenges associated with generating uniformly growing, large, quasi-two-dimensional bacterial assemblies by a membrane-based microfluidic device and report the emergence of glassy states in two-dimensional suspension of Escherichia coli . As the number density increases by cell growth, populations of motile bacteria transition to a glassy state, where cells are packed and unable to move. This takes place in two steps, the first one suppressing only the orientational modes and the second one vitrifying the motion completely. Characterizing each phase through statistical analyses and investigations of individual motion of bacteria, we find not only characteristic features of glass such as rapid slowdown, dynamic heterogeneity, and cage effects, but also a few properties distinguished from those of thermal glass. These distinctive properties include the spontaneous formation of micro-domains of aligned cells with collective motion, the appearance of an unusual signal in the dynamic susceptibility, and the dynamic slowdown with a density dependence generally forbidden for thermal systems. Our results are expected to capture general characteristics of such active rod glass, which may serve as a physical mechanism underlying dense bacterial aggregates., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)