Little is known about the long-term tillage and cropping management effects on the microbiologically derived factors that influence macroaggregates in semi-arid soil. We tested the hypothesis that differences in macro-aggregation are due to changes in soil structure related to management treatment-induced microbiological changes. In an experiment, microbiological factors consisting of aggregate stability, glomalin, russuloid basidiomycete fungi, uronic acids, total organic C (TOC), and total N (TN) were quantified in macroaggregate-size classes ranging from 4.75 to 0.25 mm, collected at 0-5 cm depth for the following treatments: (1) 12th year of fallow phase after 11 years of conventional- and no-tilled spring wheat-fallow (CTF and NTF), (2) 12th year of lentil phase after 11 years of conventional- and no-tilled spring wheat-lentil (CTL and NTL), (3) 12 years no-tilled continuous spring wheat (NTCW), and (4) 16 years uncultivated pasture (P) used as a baseline treatment. Immunoreactive easily extractable glomalin concentration was five to six times greater under P, NTCW, or NTL in the 2.00-1.00- and 1.00-0.50-mm macroaggregate-size classes than the other treatments and these results corroborated well with the results from aggregate stability assays. Russuloid basidiomycetes were highest in all NTCW macroaggregate-size classes, suggesting that annual input of lignin-containing wheat residues may influence the growth and survival of these fungi. Uronic acid amounts were highest in P but did not differ among the other treatments. In all macroaggregate-size classes, TOC content was greater in NTCW compared to CTF, and TN was about three times higher in NTL than NTF or CTF. In conclusion, 12 years of NTCW management in semi-arid soil has resulted in higher macroaggregate stability, glomalin concentration, russuloid basidiomycete populations, and TOC in macroaggregates compared to alternate-year fallow. Lentil can be used to replace fallow in dryland wheat rotation under no-till to enhance TN content and improve soil macro-aggregation.