Management of stream nutrients is becoming increasingly important in order to protect both water quality and aquatic resources throughout the USA. Using an extensive water quality database from the long-term Maryland Biological Stream Survey (MBSS), we describe nutrient relationships to landscape characteristics as total nitrogen (TN) and total phosphorus (TP) of small-order, non-tidal streams in USEPA L2 and L3 ecoregions in Maryland and by MBSS stream order at the L2 and L3 ecoregion levels. To protect stream ecosystem integrity, preliminary reference nutrient estimates (TN and TP) as percentiles (25th of all stream reaches and 75th of stream reference reaches) for the six Maryland L3 ecoregions are: Blue Ridge TN 0.29 and 0.64 mg/L, TP 0.0065 and 0.0090 mg/L; Central Appalachians TN 0.40 and 1.0 mg/L, TP 0.0060 and 0.015 mg/L; Middle Atlantic Coastal Plains TN 0.93 and 2.5 mg/L, TP 0.094 and 0.065 mg/L; Northern Piedmont TN 1.6 and 1.8 mg/L, TP 0.010 and 0.015 mg/L; Ridge and Valley TN 0.40 and 0.98 mg/L, TP 0.0063 and 0.012 mg/L; and Southeastern Plains TN 0.33 and 0.82 mg/L, TP 0.016 and 0.042 mg/L. High levels of both TN and TP are present in many streams found in non-tidal watersheds associated with all Maryland ecoregions, but are especially elevated in the Northern Piedmont and Middle Atlantic Coastal Plain ecoregions, with the latter second-order streams (average TN > 2.9 mg/L) significantly higher than all other ecoregion-order combinations. Across all six ecoregions, mean nutrient loading for both TN and TP was generally equivalent in first-order streams to nutrient concentrations seen in both second- and third-order streams, indicating a definite need to increase efforts in preventing nutrients from entering first-order streams. Small-order stream nutrient levels are the drivers for subsequent TN and TP inputs into the upper freshwater tidal reaches of the Chesapeake Bay, resulting in a potential risk for altered estuarine ecosystems.