Expression of the steffimycin gene cluster in Steptomyces albus in combination with plasmids directing the biosynthesis of different neutral and branched-chain deoxyhexoses led to the identification of twelve new glycosylated derivatives of steffimycin with different degrees of decoration in the tetracyclic core. These experiments demonstrate the flexibility of L-rhamnosyltransferase StfG for recognition of a variety of D- and L-deoxyhexoses, harboring different degrees of deoxygenation as 2-deoxyhexoses, 2,6-deoxyhexoses, and 2,3,6-deoxyhexoses, and their attachment to 8-demethoxy-10-deoxysteffimycinone. In addition, the flexibility of 3′-O-methyltransferase OleY, from Streptomyces, for the methylation of deoxyhexoses attached to the steffimycin aglycone is shown by expression of oleY in Streptomyces steffisburgensis, leading to the isolation of 3′-O-methylsteffimycin. Analysis of the biological activities of these compounds against three human tumor cell lines—breast adenocarcinoma, non-small cell lung cancer, and colon adenocarcinoma—revealed two of them, 3′-O-methylsteffimycin and D-digitoxosyl-8-demethoxy-10-deoxysteffimycinone, to possess improved antitumor activities, showing GI50 values below 1.0 μM, while steffimycin’s GI50 values fluctuate between 2.61 to 6.79 μM depending upon the cell line used. The antitumor activity data provide some insights into the structure–activity relationships of the new steffimycin derivatives, in relation to the configuration of hydroxy groups at positions C-3′ and C-4′ of the sugar moiety and positions C-8 and C-10 of the tetracyclic core.